The invention relates to methods for quenching electrophiles.
The transmission of viral diseases (e.g., hepatitis A, B, and C, acquired immunodeficiency syndrome, and cytomegalovirus infections) by blood or blood products is a significant problem in medicine. Screening donor blood for viral markers can help reduce the transmission of viruses to recipients, but many screening methods are directed to only a few discrete viruses and are therefore incomplete or less than 100% sensitive. Furthermore, other biological compositions, such as mammalian and hybridoma cell lines, products of cell lines, milk, colostrum, and sperm, can contain infectious viruses as well. It is therefore important to inactivate viruses contained in donor blood, blood products, or other biological compositions.
A number of agents that are capable of inactivating viruses in blood have been developed. For example, ethyleneimine monomer and ethyleneimine oligomers are very effective viral inactivating agents. These agents are themselves toxic, and must therefore be rendered non-toxic before a product, such as blood, is used clinically. Typically, a viral inactivating compound, such as ethyleneimine dimer, is added to a biological composition to inactivate infectious viruses that might be present in the composition. A quenching agent is then added to inactivate the ethyleneimine dimer that remains after viral inactivation has taken place. The end result is a biological composition that is relatively free of infectious viruses, but that is contaminated with quenched inactivating agent and with quenching agent.
In one aspect, the invention features a method of inactivating a contaminant, such as a virus, of a biological matrix; the method includes the steps of: (a) contacting the biological matrix with an inactivating agent including an aziridino moiety, where a portion of the agent reacts with and inactivates the contaminant, and a portion of the agent remains unreacted; (b) contacting the product of step (a) with a solid support including at least 1 quenching moiety attached to the solid support through covalent bonds, under conditions and for a time sufficient to allow the unreacted agent to bond covalently to the quenching moiety; and (c) separating the solid support and the unreacted agent from the biological matrix, where the unreacted agent is attached to the solid support through covalent bonds.
A preferred quenching moiety includes a nucleophilic moiety, such as a thiophosphate group; the thiophosphate group may be part of an intemucleotide linkage of a oligonucleotide sequence. Preferably, the solid support contains at least 2 mmol/g thiophosphate moieties.
The inactivating agent may be, for example, ethyleneimine or an oligomer of ethyleneimine. The biological matrix may be selected from the group consisting of whole mammalian blood, purified or partially purified blood proteins, blood cell proteins, milk, saliva, blood plasma, platelet-rich plasma, a plasma concentrate, a precipitate from any fractionation of plasma, a supernatant from any fractionation of plasma, a serum, a cryoprecipitate, a cryosupernatant, a cell lysate, a mammalian cell culture, a mammalian culture medium, a placental extract, a product of fermentation, a red blood cell concentrate, a platelet concentrate, a leukocyte concentrate, semen, red blood cells, and a recombinant protein-containing composition produced in a transgenic mammal. Preferably, the biological matrix is whole human blood or human blood plasma.
In a second aspect, the invention features a method of inactivating a contaminant of a biological matrix; the method includes the steps of: (a) contacting the biological matrix with an inactivating agent including an aziridino moiety, where a portion of the agent reacts with and inactivates the contaminant, and a portion of the agent remains unreacted; (b) contacting the product of step (a) with a quenching agent under conditions and for a time sufficient to allow the quenching agent to quench and to bond covalently to the unreacted inactivating agent; (c) contacting the product of step (b) with a solid support including at least 1 aldehyde moiety covalently bonded to the solid support, under conditions and for a time sufficient to allow the quenched inactivating agent to bond covalently to the aldehyde moiety; and (d) separating (i) the solid support, (ii) the quenched inactivating agent, and (iii) the quenching agent from the biological matrix, where the inactivating agent and the quenching agent are attached to the solid support through covalent bonds.
Preferred quenching agents include thiophosphate moieties and thiosulfate moieties. Preferably, the solid support contains at least 2 mmol/g aldehyde moieties attached to it.
In a third aspect, the invention features a method of quenching an electrophile; the method includes contacting the electrophile with a solid support including at least 1 thiophosphate moiety attached to the solid support through covalent bonds, under conditions and for a time sufficient to allow the electrophile to bond covalently to the thiophosphate moiety. The solid support preferably includes at least 2 mmol/g thiophosphate moieties, and more preferably includes at least 100 mmol/g thiophosphate moieties. In preferred methods, a plurality of the thiophosphate moieties are substituted with at least one C1-12 saturated or unsaturated hydrocarbon skeleton that is unsubstituted or has between 1 and 4, inclusive, substituents, independently selected from the group consisting of hydroxyl, amino, cyano, and azido.
Preferably, the electrophile includes an aziridino moiety. For example, the electrophile may be ethyleneimine or an oligomer of ethyleneimine.
In a fourth aspect, the invention features a method of removing a viral inactivating agent from a biological matrix; the method includes the steps of: (a) contacting the inactivating agent with a quenching agent that is attached to a solid support through covalent bonds; and (b) removing the inactivating agent, the quenching agent, and the solid support from the biological matrix. Preferably, step (a) includes contacting the inactivating agent with the quenching agent under conditions and for a time sufficient to allow covalent bonds to form between the inactivating agent and the quenching agent. A preferred quenching agent includes a nucleophilic moiety, such as a thiophosphate moiety.
By xe2x80x9cquenching moietyxe2x80x9d or xe2x80x9cquenching agentxe2x80x9d is meant a moiety or an agent that is capable of reacting with, and thereby reducing the reactivity of, an electrophilic compound.
By xe2x80x9cbiological matrixxe2x80x9d is meant a biological composition that contains biological macromolecules, such as proteins, nucleic acids, lipids, and carbohydrates.
The invention provides methods for quenching inactivating agents in a biological matrix. The resulting biological matrix is substantially free of infectious viruses; in addition, it does not contain remnants of the quenching agent or the quenched inactivating agent. The invention therefore provides purer products than can be obtained using conventional quenching techniques.
Other features and advantages of the invention will be apparent from the following description and from the claims.